This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have continued at the structural analysis of mitochondrial cytochrome c oxidase to elucidate its proton pumping mechanism. We are aiming to collect the datasets better than 1.4 Angstrom resolution to determine hydrogen atom position. We have observed diffraction spots at 1.31 and 1.52 Angstrom resolutions from two types of crystals under conventional cryocooling, however, soaking of the crystals in cryoprotectant increase mosaicity. To improve the mosaicity and resolution, we will attempt the high-pressure cryocooling in collaboration with Dr. Sol. Gruner's group at Cornell. If we use CHESS beamline, we can see the result right after the treatment and modify it. Therefore, we are requesting 2 day beamtime and if successful, collecting full datasets.